Carbamylation of Proteins in 2-D ElectrophoresisMyth or Reality?

Abstract

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.

Age-dependent Effects of 17β-estradiol on the Dynamics of Estrogen Receptor β (ERβ) Protein–Protein Interactions in the Ventral Hippocampus

“…Moreover, S-nitrosylation of interaction partners mediated through ER␤ could result in the characteristic "chain" patterns observed in the 2D-DIGE experiments. Other possible explanations for the chain patterns are modifications to protein charge such as carbamylation of proteins, which can occur in urea-based buffers, or phosphorylation (51). Protein modifiers such as p38 and SUMO are reported to affect ER␤ signaling and change with age (52-54); thus it is possible that modifications to ER␤ or its interaction partners by these types of proteins could contribute to the observed effects.…”

Identification and characterization of a novel isoform of the vesicular γ-aminobutyric acid transporter with glucose-regulated expression in rat islets

“…To enhance the solubility of membrane proteins, islet samples were prepared in SDS boiling buffer (Ames & Nikaido 1976). No urea was added to the sample buffer, and samples were kept frozen at 80 C, avoiding any risk of carbamylation (McCarthy et al 2003). Isoelectric focusing was carried out in glass tubes using 2% pH 3·5-10 ampholines (Amersham).…”

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

“…Firstly the chemical properties of the UTS buffer, this solution must not be overheated, or carbamylation occurs on primary amines and can therefore interfere with both cyanine dye coupling and 2D profile, worsening spots resolution by impairing IEF 15 . Secondly, the available pH ranges, nowadays the widest pH interval is among 3 to 11, this factor joined to the probability that not all proteins entry onto the IPG strip may explain the stacked proteins bands in the both sides of the gels after staining.…”